Diversity arrays technology DArT markers in appl

Authors: Henk J Schouten W Eric van de Weg Jason Carling Sabaz Ali Khan Steven J McKay Martijn P W van Kaauwen Alexander H J Wittenberg Herma J J Koehorstvan Putten Yolanda Noordijk Zhongshan Gao D Jasper G Rees Maria M Van Dyk Damian Jaccoud Michael J Considine Andrzej Kilian

Publish Date: 2011/05/15

Volume: 29, Issue: 3, Pages: 645-660

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Abstract

Diversity Arrays Technology DArT provides a highthroughput wholegenome genotyping platform for the detection and scoring of hundreds of polymorphic loci without any need for prior sequence information The work presented here details the development and performance of a DArT genotyping array for apple This is the first paper on DArT in horticultural trees Genetic mapping of DArT markers in two mapping populations and their integration with other marker types showed that DArT is a powerful highthroughput method for obtaining accurate and reproducible marker data despite the low cost per data point This method appears to be suitable for aligning the genetic maps of different segregating populations The standard complexity reduction method based on the methylationsensitive PstI restriction enzyme resulted in a high frequency of markers although there was 52–54 redundancy due to the repeated sampling of highly similar sequences Sequencing of the marker clones showed that they are significantly enriched for lowcopy genic regions The genome coverage using the standard method was 55–76 For improved genome coverage an alternative complexity reduction method was examined which resulted in less redundancy and additional segregating markers The DArT markers proved to be of high quality and were very suitable for genetic mapping at low cost for the apple providing moderate genome coverageThe genus Malus has been a focus of molecular studies since the mid1980s when Weeden and Lamb 1985 used isozymes as a means of discriminating between different apple cultivars As new marker types have been developed scientists have readily adopted them into their studies of apple genetics progressing from rapid amplification of polymorphic DNA RAPD Koller et al 1993 through restriction fragment length polymorphism RFLP and isozymes Maliepaard et al 1998 to amplified fragment length polymorphism AFLP and simple sequence repeat SSR Guilford et al 1997 Hokanson et al 1998 and on to targeted markers eg Broothaerts 2003 Calenge et al 2005 Chagné et al 2007 and single nucleotide polymorphism SNP arrays Micheletti et al 2011 The progress in the molecular characterisation of the Malus genome has recently gained further momentum with wholegenome sequencing Velasco et al 2010Molecular markers have been applied widely to evolutionary and pedigree studies in apple including both wild Malus species Richards et al 2009 and domestic cultivars eg Cabe et al 2005 Evans et al 2010 Additionally markers developed for the apple have been applied fairly widely to other pome species and vice versa notably pear Pyrus spp Yamamoto et al 2001 Hemmat et al 2003 Dondini et al 2004 quince Cydonia oblonga Yamamoto et al 2004 and loquat Eriobotrya japonica Gisbert et al 2009 He et al 2010 Simultaneously linkage maps have been constructed for a number of domestic cultivars and several map alignments have been reported Maliepaard et al 1998 N’Diaye et al 2008 Patocchi et al 2009 Van Dyk et al 2010 The construction of linkage maps has facilitated the identification of molecular markers associated with numerous phenotypic traits Among the traits examined to date are resistance to apple scab caused by the fungus Venturia inaequalis Koller et al 1994 Calenge et al 2004 Bus et al 2005a b Soriano et al 2009 fire blight caused by the bacterium Erwinia amylovora Peil et al 2007 columnar growth habit Moriya et al 2009 several fruit quality traits eg King et al 2001 Liebhard et al 2003a Costa et al 2005 2008 2010 Kenis et al 2008 and chilling requirement Van Dyk et al 2010 The identification of such molecular markers is essential for markerassisted selection in apple breeding programs Gianfranceschi et al 1996 Liebhard et al 2003b Gardiner et al 2007 Zhu and Barrett 2008 In recent years genomic methods have been embraced by apple researchers The enhanced ability to study gene expression has resulted in new understandings of developmental processes Ban et al 2007 Espley et al 2009 Transcription analyses of apple fruit development using cDNA microarrays Soglio et al 2009 and plant physiological responses to pathogens Norelli et al 2009 has facilitated the development of new molecular markers eg Chagné et al 2008 Igarashi et al 2008 The further development of highthroughput genetic technologies will continue to expand the ability of scientists to investigate the details of the genetics of apple and its relatives Shulaev et al 2008Since the proofofconcept paper Jaccoud et al 2001 Diversity Arrays Technology DarT has been developed as an inexpensive wholegenome profiling technique for many organisms especially plants The website wwwdiversityarrayscom has a current list of organisms for which arrays are available 50 DArT in its current implementation is a hybridisationbased genome profiling technology that does not require sequence information and uses microarrays to identify and type DNA polymorphisms As the DArT markers are typed in parallel it is possible to identify hundreds or even thousands of polymorphic markers in a single experiment Wittenberg et al 2009 This highly parallel assay results in a reduction of the price per data point to around US 001 in organisms with welldeveloped arrays The DArT assay primarily detects dominant markers mostly resulting from single nucleotide polymorphisms and indels in restriction sites and differences in methylation of restriction sites When methylationsensitive restriction enzymes like PstI see Gruenbaum et al 1981 were used in the large genomes of cereals Wenzl et al 2004 2006 Akbari et al 2006 DArT markers were located preferentially at the generich subtelomeric regions of the chromosomes The use of methylationsensitive enzymes may provide also an insight into epigenetic variation Wenzl et al 2004Interestingly while DArT has performed well in over 50 crops there are no reported applications of DArT in horticultural trees As DArT has been applied successfully to complex amphidiploid genomes like wheat Akbari et al 2006 oat Tinker et al 2009 and sugarcane HellerUszynska et al 2010 application to the duplicated genomes of the pome fruits such as apple Velasco et al 2010 pear and loquat should be promising too Here we present the development and validation of DArT for apple using a complexity reduction method similar to the one used for the cereal genomes We compare this with a second complexity reduction method that is similar to the method used for the fungus Mycosphaerella graminicola Wittenberg et al 2009 and we discuss the characteristics of the detected markers We combined DArT markers with other marker types in genetic linkage maps providing insight into the coverage of the DArT markers in the apple genome We used as a starting point the progeny and genetic linkage map of Prima × Fiesta which was the first genetic linkage map for apple covering all 17 chromosomes Maliepaard et al 1998 In addition we used a more recent progeny of other parents for genetic mapping Furthermore we evaluated the performance of DArT in a genetic diversity analysis of 44 diverse apple accessions and a set of Australian breeding linesFor the building of the DArT libraries care was taken to represent a wide genetic diversity including several major founders in apple breeding worldwide founders of more local breeding programs modern cultivars and some very recent selections from ongoing breeding programs Fortyfour accessions of Malus were used for the library development Online Resource 1a

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